Transcriptional regulation plays an important role in cell differentiation and in maintaining cell phenotypes, and misregulation often leads to disease. In eukaryotes, chromatin is maintained in a repressive state by the nucleosomal structure. Consequently, transcription activation requires creation of nucleosome free regions for regulatory factor binding. Similarly, transcription elongation requires destabilization of the nucleosomes ahead of the transcribing machinery. The mechanism by which the nucleosome structure inhibits regulatory factor binding has, however, not been fully elucidated. Current knowledge from biophysical experiments challenges the possibility of limited access as the mechanism of inhibition. This is especially obvious considering that the DNA winds on the exterior surface of the core histone octamer being continuously exposed toward the surroundings. Further, it has been shown that even regions of dense chromatin packaging are freely accessible to relatively large ligands partly due to the relatively large pore-size in the chromatin matrix and to the local Brownian motion occasionally bringing sites buried in chromatin domains to the surface where large TF would bind. Therefore, the question how access to DNA in chromatin is regulated remains unanswered. I have considered the possible role of alteration of DNA structure that results from packaging of DNA into chromatin as a mechanism of controlled access. This is bearing in mind the important contribution that DNA shape makes to TF binding specificity. Further, it is well known that TF binding to DNA results in deformation of both the DNA and protein so as to form a proper fit between the macromolecules. However, such a plasticity may not be allowed in view of the limited degrees of freedom that DNA in chromatin has. In my work, I evaluated the binding of fluorescent, small molecule intercalators to DNA in situ. Intercalation requires deformation of DNA akin to that of TF binding. Given their low molecular weight, small molecules would be expected to easily access the DNA in all chromatin domains and there intercalation could be easily quantified from their fluorescence. My results revealed that intercalation was limited to extranucleosomal DNA, while intercalation into the nucleosomal DNA only became possible after histone eviction. Intercalation into extranucleosomal DNA could be moderately enhanced by topological relaxation in the wake of nicking the DNA. A single nick per 50 kbp loop was sufficient to achieve this increment revealing that the conformational change elicited readily spreads along the chromatin. Staining with DAPI, which binds to the minor groove without intercalation, was unimpeded by the presence of nucleosomes. These data have led me to conclude that the conformational constraint imposed on DNA by the several chemical bonds linking the DNA to the nucleosome core are likely responsible for this shielding of the DNA to particular small molecules from inside, while the internucleosomal DNA can adapt to bind the ligand. Next, I evaluated the effect of supercoil relaxation on the binding of HMGB1, a protein whose binding to DNA partly involves intercalation. These measurements have yielded a complex picture. Relaxation of supercoiling by using DNA nicking agents had no effect of HMGB1 binding in vivo. However, binding of Dox, a membrane permeable DNA intercalator, led to an initial increase in HMGB1 binding in vivo, followed by decreased binding at higher concentrations. In solution, intercalator binding caused a monotonous decrease in HMGB1 binding to supercoiled plasmid DNA. Having assessed also the effect of Dox on the binding of the linker histone H1, known to antagonize HMGB1 binding, has led me to conclude that Dox used at small concentrations enhances HMGB1 binding by destabilizing H1 binding, while in the higher concentration range competes with HMGB1 for the DNA. Nucleosomes emerge as major impediments of ligand binding as a result of the constraint of DNA conformation, while the extranucleosomal DNA exhibits a higher degree of conformational plasticity, allowing for a complexity of molecular interactions. Since the conformational features determining intercalation are tightly interdependent with all the other DNA conformational features, based on the powerful effect of nucleosomal constraint on intercalator binding I anticipate an analogous effect of the nucleosomal structure on any ligand binding, including that of TFs. My data also suggests that anthracycline therapy may be synergized by chemotherapeutic agents that induce DNA breaks such as topoisomerase inhibitors and bleomycin, or radiotherapy.